Poly (ADP-ribose) polymerase inhibitors (PARPis) have clinical promise, but their potential has yet to be fully realized. These drugs have shown efficacy in hereditary breast cancers with defects in BRCA1/2 that participate in a particular form of DNA double strand break (DSB) repair, homologous recombination (HR). However, PARPi studies in sporadic triple negative breast cancers and other tumor types have been disappointing. We have recently shown that an alternative and highly error-prone pathway that involves PARP- 1 is significantly increased in multiple sporadic cancers, including triple negative breast cancer and leukemia (chronic and acute myeloid leukemia [CML and AML]). The central goal of this application is to test the novel hypothesis that sensitivity to PARP inhibitors is dependent, in part, on ALT NHEJ. Studies in Aim 1 will determine the extent to which sensitivity to PARPis is dependent on ALT NHEJ activity in breast and leukemia cell lines that have already been demonstrated with high ALT NHEJ, compared with immortalized non-tumorigenic cells in which ALT NHEJ is low. The first part of this aim will focus on the effects of PARPis on ALT NHEJ activity, using state of the art assays that measure ALT NHEJ. The second part of this aim will determine the extent to which ALT NHEJ activity is required for cell survival. Studie in Aim 2 will focus on AML, for which novel therapies are urgently required. We will determine the relationship between ALT NHEJ and sensitivity to PARPis in primary AML cells using in vitro assays and innovative bioluminescence-based xenograft models of AML in vivo. These studies may shed further light on the molecular mechanisms underlying sensitivity to PARPis. Moreover, these studies may uncover potential novel biomarker concepts for predicting those cancer patients most likely to benefit from PARPis, hopefully provide a compelling rationale for the development of this novel therapeutic strategy for sporadic cancers with high ALT NHEJ activity.